Small-molecule combinations expand hematopoietic stem cell ex vivo and in vivo Abstract Hematopoietic stem cells (HSCs) are featured by their self-renewal potential and ability to differentiate into multiple blood lineages. Hematopoietic stem cell transplant (HSCT) is a mainstay of life-saving therapy for hematopoietic malignancies and hypoproliferative disorders. The use of noninvasively accessible umbilical cord blood (UCB)-derived HSCs provides many advantages over bone marrow and peripheral blood HSC, including less stringent requirement for human leukocyte antigen (HLA) match, decreased risk of graft-versus- host disease (GVHD), etc. However, limited cell dose in a UCB unit may lead to significant problems such as delayed engraftment, engraftment failure, and severe infectious complications. The expansion of HSCs from UCB has remained an important goal to develop advanced cell therapies for enhancing transplantation efficacy and providing potential cure for various hematological diseases. Recent studies have achieved ex vivo expansion of HSCs using cytokine cocktails combined with small molecules, including pyrimidoindole derivative (UM171), aryl hydrocarbon receptor antagonist (SR1). The major disadvantage with these approaches is that it is only effective in the presence of high concentration of cytokines. Cytokines have been known to induce differentiation and affect the function of primitive HSC, they might have long-term deleterious effect on humans due to skewed reconstitution of the hematopoietic system. The small-molecule combinations with minimal cytokines would be ideal for clinical applications. Our published studies demonstrated a combination of two inhibitors (2i) allows the ex vivo maintenance of human and mouse long-term HSCs and doing so under cytokine-free condition. The combination of two clinical drugs (lithium plus rapamycin) leads to increase of long-term mouse HSCs in vivo. In our recent studies, we found that 2i works synergistically with SR1 under low cytokine condition, significantly expanded long-term functional HSC ex vivo. In this project, we will carry out studies to optimize culture condition of small-molecule combination regimens. Furthermore, we will test and optimize the in vivo expansion method, which will greatly improve the successful rate of HSC transplantation. Our major goal is to develop new clinical protocols for expanding functional UCB- derived HSCs for therapeutic applications.